Manufacture of serums from animals, excluding man



. J. PENFOLD.

MANUFACTURE OF SERUMS FROM A'NIMALS, EXCLUDING MAN.

APPLICATION FILED SEPT 14. I920.

Patehted May 24, 1921.

WILLIAM JAMES PENFOLD, or nnonswrcx, vrcronm, nusmm MANUFACTURE OI BERUMS FROM ANIMALS, EXCLUDING MAN,

Specification of Letters Patent.

Patented May 24, 1921.

App1ieat1on filed September 14, 1920. Serial No. 410,204.

To allwkomitmay concern: 4

Be it known that I, WILLIAM JAMES PEN- FOLD, a subject of the K'in of Great Britain,

residing at Park street, runswick, in the State of Victoria, Commonwealth of Australia, bacteriologist, have invented certain. new and useful Improvements in the Manu facture of Serums from Animals, Excluding Man, of which the following is a specification.

This invention relates to certain improvements in the manufacture of serums from animals, excluding man, and refers more especially to the production of serums which are obtained from animals which have been rendered immune, to bacteria or their toxins. This invention, however, is not confined to the production of anti-toxic or anti-bacterial serums but may be applied to the production of other serums. v

In the production of serums as at present practised it is customary to extract the blood from an animal (generally the horse) which has or has not been rendered immune and this blood is treated for the separation of the serum therefrom.

The separation of the serum is customarlly effected ineither of several ways:

1. By receiving the blood into a jar or flask in ,which it is allowed to clot and then expressing the serum therefrom by suitable pressure. In this method the red cells of the'blood are permanently entangled in the clot. 2. By receiving the blood into bottles or flasks containing a solution of oxalate or sodium citrate or some other anti-clotting agent, and allowing the blood to stand therein so that its cellular elements sediment and the supernatant plasma can be removed into other containers' This plasma is then clotted by the addition of a lime salt and the serum is expressed from the clotted plasma by suitable pressure. I

3. By receiving the blood into containers coated with parafiin or some other like substance which prevents clotting, allowing the cellular elements to settle, withdrawing the supernatant plasma, which is then clotted, and subsequently separating the serum from the clot.

All these methods however, have the rave disadvantage that they produce proound anemia, so that after a first bleeding of eight to ten liters'in the case of a horse,

for example, a weeks interval should be allowed before a second bleedin is unde taken. This weeks interval is li y no mearis SllfllClGIlt to allow of complete recover from the anem a. A horse may be bled 1n this way each week, but if the bleeding be to the extent above indicated the animal becomes progressively more anemic, so that after two, three or four bleedings, it is necessary to give it a prolonged rest. This rest usually consists of one week for every individual bleeding, that is to say, if the horse has been bled every week for four weeks, it should be given one months rest. In some laboratories fairly large bleedings are continued weekly, but horses so treated get out of condition and do not live long.

The object of this invention is to provide a method of procedure whereby this anemia is removed forthwith, thereby allowing of the more frequent and profuse bleeding of the horses or other animals.

I have found that the red cells of the blood, after the separation of the plasma therefrom, may be introduced into the blood vessels of an animal from which the blood had been previously removed. These red cells are absorbed in the system of the animal together with the small quantity of plasma associated with them and the anemia to WhlCh the animal is subject is thereby removed.

This invention comprises therefore the returning and the means for returning the red cells of the blood, after the separation of the plasma, into the blood vessels of the animal so as to remove its anemia and increase its serum yielding capacity.

This invention is applicable to those methods in vogue as above indicated, in whlch the cellular elements of the blood are separated from the plasma, but is not applicable to the first-described method in which the red cells become permanently entangled in the clot.

In carrying out my invention to obtain 1 serums from the horse I proceed as folblood, by agitation, as the blood is running from the vein into the receiver. The bottles are then permitted to stand to allow of sedimentation of the red cells. This separation may be accelerated by the use of a centrifuge, but in the case of horse blood the centrifuge isnot necessary. After the red cells have sedimented, the supernatant plasma is removed into one or more suitable containers, and the remaining red cell fractions are collected into other containers known as returning bottles. These red cells by suitable means are re-injected into the vein of the horse.

In order that the invention maybe the better understood, reference may be made to the accompanying sheet of drawings, which illustrate the apparatus which may be used in carrying out my method, but it should be understood that I do not propose shading, and the supernatant plasma by lighter shading.

Fig. 3 shows the same receiving bottle, containing the red cells, connected with another bottle, into which the red cells are delivered through a suitable filter.

Fig. 4' shows this last-mentioned bottle into which the filtered red cells have been received, connected with the cannula, through which the red cells are returned into the blood vessels, of the animal.

The apparatus illustrated in thesedraw-' ings comprises three bottles or containers (or series thereof) viz., a receiving or bleeding bottle A, a plasma bottle B, and a returning bottle C. In commencing o erations the receiving or bleeding bottle is provided with a stopper A having two holes, through one of which passes a glass tube A connected by a rubber tube A to a cannula D. The otherhole in the stopper A" is fitted with a glass tube A having a bulb containing cotton wool-to act as a-filter and to-mainture of the blood with the anti-clotting agent tam sterile conditions.

The cannula D is inserted into the vein of the animal (saya horse) and the blood is withdrawn into the bleeding bottle A (Fig. 1) within which a. small amount of potassium oxalate solution (or other anti clotting agent) has been placed. The bottle A is continuously agitated while the blood isrunning into it, whereby thorough admixis secured. When the bleeding is completed the cannula D is removed'from the vein of the horse and is disconnected from the tube A leading into thebottle which tube is then closed by means of a plug of cotton wool and leadfoil cap. Afterthe sedimentation of the red cells has taken place (as shown in darker shading in 2') the stopper A is removed from the bottlev A" and another stopper. A is inserted therein having a downwardl projecting tube A which may be adjuste in height for withdrawin the supernatant lasma leaving the redicel' s undisturbed. he supernatant plasma (as shown in lighter shading in Fig, 2 is then passed by means of pressure oif suction through the tube A into thebottle B. A

bottle B for thepurpose of clotting the same lime salt is thenadded to the plasma in the q and the serum may be separated therein or more conveniently the plasma may be transferred toa separate jar m which the serum is expressed from the clot in the usual way. Another stopper A is now inserted into the bottle A having a tube A which passes down to the bottom of the bottle for the re moval of the red cells. This'tube A is conturnin bottle C having amuslin filter C throug which the red cells are filtered. The pressure within the bottle A being raised, the red cells are then transferred into the bottle C, or alternatively the pressurewithin the bottle C may be diminished, and in,

this way the red'cells are all sucked into the returning bottle C. j The bottle A is then disconnected from the bottle 0, the connect? ing tube C is closed .by means'of a stop co'ck G and the exit tube C -is connected with the canula D. Pressure is then applied to at nected with. a tube .C-' 7 leading into the. re

the bottleC'by bellows C whereby. the red cells are forced into the canula D, which is inserted into the vein of the animal, and these red 'cells are returned to the blood vessels of the animal.

It is not necessary to add any diluting fluid to the red cells as therev is a slight amount of plasma left around them which is suflicient to enable this'red cell fraction to flow back into the animals vein.

The sedimentation of the red cells in the case of horse blood occurs more quickly if it is allowed, to takeplace at a warm temperature, and since it is advantageous to have the red cells 'at the temperature of the animal body when returning them, it

is desirable to have the bottles standin dur- .ing the sedimentation process at bloo heathigher. temperature.

or a slightly g system I can take thirty-six. liters in the course of five days, without produc 1 of blood in' threebleedings out ofahorse" ing the prolonged anemla which-the extraction of ten liters of blood previously pro- I duced. The degree ofanemia in the horse I has been judged by hemoglobin pe estimations; These liters of plasma, whereas by the ordinary method S-Iiters of blood yield about 5.2

red. cell'counts and i 36 liters-of blood yield slightly-over 22.5

liters of plasma, that is, 22.5 liters of plasma i are obtained in five days in teed of about.

' of blood in five days, the percentage fall of.

5.2 liters in seven days. If 10 liters of blood be taken from a horse in the ordinary way and nothing returned to the horse, the

percentage fall of red cells is equal to about 30% depending on the size of the horse.

Under my system, after removing 36 liters red cells observed after the return of the red cells of the third bleeding is negligible. The protein constituents of the plasma are not seriously-diminished by these. massive bleedings. It has been ascertained that a percentage fall from 7.8% to 7% occurred after 36 liters of blood had been extracted in five days, whereas under the old system a similar fall may be found after the removal of 10 liters.

Instead of returning the red cells into the blood vessels of the animal whence they came, it will be obvious that a similar result may be obtained by transfusion of the red cells obtained from one animal (after extraction of the plasma) into the blood vessels of another animal of the same species which has been bled.

I claim- 1 In the manufacture of serums from animals (excluding man) the returning of the red cells of the blood, afterseparation from the plasma, into the blood-vessels of the animal.

2. An improved method of serum production consisting of bleeding the animal (excluding man) under such conditions that clotting of the blood is prevented, separating the' supernatant plasma from the red cellular elements, separating the serum from the plasma, and returning the red cells to the blood vessels of the animal.

3. An improved method of serum production consistin of receiving the blood from the animal excluding man) into vessels containing an anti-clotting agent (such as oxalate solution) allowing the blood to stand until the red cells sediment, separating the supernatant plasma, adding a, clotting agent (such as a lime salt) to the plasma, separating the serum from the plasma, filtering the red cells and returning the red cells to the blood vessels of the animal substantially as described.

In testimony, that I claim the foregoing as my inventlon, I have signed my name in presence of two subscribin witnesses.

WILLIAM JAMES ENFOLD. Witnesses: G. R. GULLER,

BERTHA R. CLARKE. 

